Sandwich ELISA for Measurement of Cytosolic Aspartate Aminotransferase in Sera from Patients with Liver Diseases

[摘要] With the introduction of modern biochemical techniques, more than 50 enzymes have been developed for diagnostics in liver tests. Among the enzymes, aspartate aminotransferase (AST; EC 2.6.1.1; formerly known as GOT) was introduced in the mid-1950s and has remained the mainstay of enzyme diagnosis for liver disease for more than half a century (1). AST is widely distributed among human organs as cytosolic and mitochondrial isotypes (2). Increased activity of this enzyme is considered one of the most sensitive indicators of hepatocellular damage (3). Previously, several attempts were made to develop an immunoassay procedure for measuring AST mass rather than enzyme activity, but they are currently not used for the diagnosis of liver diseases (4)(5)(6)(7)(8). In this study, we generated monoclonal antibodies (mAbs) to AST and used them to develop a sandwich ELISA to measure the enzyme mass in sera.Because the amino acid sequences of the human and porcine enzymes are similar, we used porcine AST as an immunogen for the production of mAbs (9). The porcine enzyme is commercially available as a highly purified form in large quantities. Twenty hybridoma cells to porcine AST were initially screened by ELISA from several fusions, and four clones were selected for further characterization.To confirm the specificity of the mAbs to the human enzyme, we immunoblotted partially purified human enzyme and total proteins extracted from a human liver with the mAbs. The antibodies specifically recognized a single protein band of 45 kDa, which coincided well with the expected size of AST. To evaluate whether the mAbs recognized a cytosolic or a mitochondrial form of AST in human tissues, we prepared cytosolic …

[学科分类] 过敏症与临床免疫学